A New Method for The Disruption of Cell Walls of Gram-Positive Bacteria and Mycobacteria On The Point of Nucleic Acid Extraction: Sand Method

Abstract

Nowadays molecular methods are widely used in the rapid diagnosis of infectious agents. Polymerase chain reaction (PCR) is the most preferred method for this purpose. Obtaining sufficient and pure DNA or RNA is important for the PCR. Different DNA extraction protocols such as phenol-chloroform, proteinase K, glass beads and boiling have been used successfully for DNA isolation from gram-negative bacteria. However since gram-positive bacteria have a thicker layer of peptidoglycan and mycobacteria have complex glycolipids in their cell walls, for the isolation of DNA or RNA from these microorganisms, the complex cell wall structure must be eliminated. For this purpose, the bacterial cell wall must be completely or partially removed forming sferoblast using lysostaphin in the Staphylococcus genus as gram-positive bacteria and using a chemical like cetyltrimethyl ammonium bromide for the Mycobacterium genus. In this study, we planned to use sand particles for the mechanical elimination of the cell wall without any need for chemicals and we called this procedure as "sand method". For the purpose of DNA extraction, the fine-grained sand was washed with ddH(2)O without losing small particles and then sterilized by autoclaving. For the purpose of RNA extraction; the sand was washed with ddH(2)O, incubated for 30 minutes with 10% HCl, and then autoclaved. A methicillin-resistant Staphylococcus aureus (MRSA) strain previously isolated and identified from a clinical specimen was mixed in 100 mu l Tris-EDTA buffer with 100 mg sand. The mixture of bacteria and sand was vortexed at the maximum speed for 5 minutes. The MRSA-sand mix was treated with proteinase K and phenol-chloroform, and ethanol precipitation protocol was then followed for obtaining DNA. For comparison of the sand method with the other methods, the same amount of bacteria used in the sand method was incubated for one hour with lysostaphin, and then the proteinase K DNA extraction method were completed in the same way used in the sand method. For obtaining RNA from M.tuberculosis H37Rv ATCC 25618, M.tuberculosis H37Ra ATCC 25177 and M.tuberculosis H37Rv Pasteur Institute RSKK 598 standard strains, bacteria were dissolved in 20 mu l Tris-EDTA buffer with 100 mg sand. The mixture of bacteria and sand was vortexed at the maximum speed for 5 minutes. After that, the classic RNA extraction protocol using guanidinium thiocyanate-phenol-chloroform (GTPC) was completed. To investigate the usefulness of the obtained DNA, a PCR was performed with specific primers for staphylokinase and enterotoxin genes that were shown in the genome of the chosen MRSA strains from our previous studies. To investigate the usefulness of the obtained RNA from the sand method; first cDNA synthesis is completed. The PCR efficiency was then tested using primers specific to the efflux pump genes of M.tuberculosis including Rv1410c, Rv2333c, and DrrA genes. To compare the effect of the sand method, GTPC protocol was applied in the same amount of mycobacteria without the sand treatment. The DNA obtained from MRSA with the application of lysostaphin and the DNA obtained from MRSA by the sand method were run in agarose gel electrophoresis. The amount and purity of DNAs were measured with a spectrophotometer. The same amount and purity of the DNAs were approximately the same in both of the extraction methods. The existence of non-inhibitors of DNA in the sand method was shown with the PCR, which have worked efficiently with the DNAs obtained from the sand method.

RNA was obtained efficiently from the Mycobacterium strains by the sand method, but no RNA could be obtained from the mycobacteria with the other methods. It was shown that the RNA obtained using the sand method worked effectively in both cDNA synthesis and PCR in which synthesized cDNA was used. The sand method described in the study worked effectively to obtain sufficient amount of pure DNA and RNA from the bacteria containing rigid cell walls that are difficult to obtain the nucleotide. It was concluded that, using the sand method instead of relatively expensive lysostaphin or other chemicals, has important advantages such as decreasing the cost and the shortening of the DNA extraction period.